Stabilizing agents for probiotic compositions

Abstract

The present invention relates to the use of surface-reacted calcium carbonate as stabilizing agent for probiotic compositions, wherein the surface-reacted calcium carbonate is natural ground calcium carbonate or precipitated calcium carbonate treated with carbon dioxide and one or more H3O + ion donors, wherein the carbon dioxide is formed in situ by H3O + ion donors and / or is supplied from an external source. The present invention relates to the use of surface-reacted calcium carbonate as stabilizing agent for probiotic compositions, wherein the surface-reacted calcium carbonate is natural ground calcium carbonate or precipitated calcium carbonate treated with carbon dioxide and one or more H3O + ion donors, wherein the carbon dioxide is formed in situ by H3O + ion donors and / or is supplied from an external source.

Description

[0001] The present invention relates to the field of probiotic agents, and in particular to the use of surface-reacted calcium carbonate as a stabilizing agent for probiotic compositions, as well as to a method for stabilizing probiotic microbial cultures, and to a method for preparing dry stabilized probiotic compositions.

[0002] Probiotics are live microorganisms that confer a health benefit on the host when administered in a sufficient amount. Microorganisms used as probiotics originate from different genera and species and have been studied for a wide variety of health and disease endpoints. Currently, both yeasts and bacteria are used as probiotics, including lactic acid bacteria, Bifidobacterium, Propionibacterium, Bacillus, and Escherichia coli. They can be naturally occurring microorganisms or microorganisms that have been genetically altered for a specific effect (see, Sanders et al., Gut Microbes 2010, 1, 3, 164-185).

[0003] Probiotics are naturally present in fermented foods, can be added to other food products, and are available as dietary supplements. Probiotics are identified by their specific strain, which includes the genus, species, subspecies if applicable, and an alphanumeric strain name. The seven core genera of microorganisms most frequently used in probiotic products are Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, Enterococcus, Escherichia, and Bacillus.

[0004] Probiotics typically exert their effects in the gastrointestinal tract, where they can influence the intestinal microbiota, the activity and composition of which can influence human health and disease. Probiotics are found to transiently colonize the human intestinal mucosa in a highly individualized pattern, depending on the baseline microbiota, probiotic strain, and region of the gastrointestinal tract.

[0005] In addition, probiotics can exert health effects through non-specific, species-specific, and strain-specific mechanisms. These mechanisms include inhibition of growth of pathogenic microorganisms in the gastrointestinal tract, for example, by promoting colonization resistance, improving intestinal transit, or helping perturbed microbiota normalize, production of bioactive metabolites, and reduction of luminal pH in the colon. Species-specific mechanisms can include vitamin synthesis, intestinal barrier strengthening, bile salt metabolism, enzymatic activity, and toxin neutralization. Strain-specific mechanisms, which are rare and used by only a few strains of a given species, include cytokine production, immunomodulation, and effects on the endocrine and nervous systems. Through all of these mechanisms, probiotics can have a wide range of effects on human health and disease.

[0006] Live microorganisms used to make many fermented foods, including yogurt, typically survive well in the product throughout its shelf life. However, they often do not survive transit through the stomach and can not resist degradation by hydrolytic enzymes and bile salts in the small intestine, and thus can not reach the distal intestine. However, compliant probiotic strains included in yogurt or other foods do survive intestinal transit.

[0007] Probiotics can also be available as dietary supplements (in capsules, powders, liquids, and other forms) containing a wide variety of strains and dosages. These products often contain mixed cultures of live microorganisms, rather than a single strain. The concentration of probiotic microorganisms in probiotic compositions is typically measured in colony forming units (CFU), which indicates the number of live cells. Many probiotic supplements contain 10 9 to 10 10 CFU per dose, but some products contain up to 5 x 10 10 CFU or more. However, higher CFU counts do not necessarily improve the health effects of the product. Because probiotics must be alive to be consumed to have health benefits and they can die during their shelf life, the CFU number at the time of manufacture is meaningless, but the CFU number at the end of the product's shelf life is meaningful (see National Institutes of Health, Probiotics, Fact Sheet for Health professionals).

[0008] Large quantities of probiotics are typically prepared by adding a stabilizer such as a polysaccharide or oligosaccharide to a concentrate from a fermentation vessel and then freezing or spray drying the concentrate, as described in US 20050100559. The dry material obtained is then ground into a powder. However, it is challenging to produce probiotic compositions that have a reasonably long shelf life, particularly at room temperature, i.e., that remain viable for at least 10 6The concentration of viable cells (colony forming units, CFU) per gram of preparation.

[0009] WO2010054439 A1 discloses a probiotic composition comprising probiotic microorganisms embedded in a matrix, wherein the matrix preserves the viability of the microorganisms.

[0010] EP3520798 A1 relates to dosage forms comprising a functionalized calcium carbonate acting as active ingredient, which are preferably used for the release of calcium. In WO2013068478 A1 carriers for the controlled release of active agents are described, which comprise a core comprising a surface-reacted natural or synthetic calcium carbonate and at least one active agent associated with the surface-reacted calcium carbonate, and a coating encapsulating the core.

[0011] However, there is still a need in the art for further methods for stabilizing probiotic microorganisms, and in particular for probiotic compositions having an extended shelf life.

[0012] It is therefore an object of the present invention to provide a stabilizing agent for probiotic compositions. It would be desirable that the stabilizing agent reduces or prevents degradation of probiotic microorganisms during drying of the probiotic composition, and / or extends the shelf life of the probiotic composition. It would also be desirable that the stabilizing agent is derivable from natural sources, is environmentally safe, and is readily degradable.

[0013] It is also an object of the present invention to provide probiotic compositions having an extended shelf life, in particular at room temperature. It would further be desirable that the probiotic composition is suitable for consumption by people having special dietary needs, such as infants, young children, elderly people or diabetics. For example, it would be desirable that the probiotic composition does not comprise sugars, polysaccharides or synthetic encapsulating materials.

[0014] According to one aspect of the present invention, the use of a surface-reacted calcium carbonate as a stabilizing agent for probiotic compositions is provided, wherein the surface-reacted calcium carbonate is a natural ground calcium carbonate or a precipitated calcium carbonate with carbon dioxide and one or more H3O + a reaction product of an ionic donor, wherein the carbon dioxide is formed in situ and / or is supplied from an external source. + The ionic donor treatment is formed in situ and / or is supplied from an external source.

[0015] According to a further aspect of the present invention, a method for stabilizing a probiotic microorganism culture is provided, comprising the following steps:

[0016] mixing a probiotic microorganism culture with a surface-reacted calcium carbonate in an aqueous medium, wherein the surface-reacted calcium carbonate is a natural ground calcium carbonate or a precipitated calcium carbonate with carbon dioxide and one or more H3O+ The reaction products of the ion donor, wherein the carbon dioxide is passed through H3O + Ion donor treatment is performed in situ and / or supplied from an external source, and

[0017] The obtained mixture is dried, wherein preferably the drying is carried out by spray drying, freeze drying, rapid drying, fluidized bed drying, jet drying, vacuum drying or a combination thereof.

[0018] According to a further aspect of the present invention, a method for preparing a dried, stabilized prebiotic composition is provided, comprising the following steps:

[0019] a) Provides an aqueous probiotic composition comprising at least 75% by weight of a probiotic microbial culture based on the total weight of the probiotic composition.

[0020] b) Provide an aqueous suspension comprising 10-30% by weight of surface-reacted calcium carbonate based on the total weight of the aqueous suspension, wherein the surface-reacted calcium carbonate is naturally milled calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid or mixtures thereof. + The reaction products of the ion donor, wherein the carbon dioxide is passed through H3O + Ion donor treatment is performed in situ and / or supplied from external sources.

[0021] The surface-reacted calcium carbonate described herein has a median particle size d of 0.1 to 75 μm. 50 And volumetric top-cutting particle size d from 0.2 to 150 μm 98 and 15m 2 / g to 200m 2 The specific surface area per g was measured using nitrogen and the BET method, and

[0022] The weight ratio of probiotic culture to surface-reacted calcium carbonate is 5:95 to 40:60.

[0023] c) Mix the probiotic composition from step a) with the surface-reacted calcium carbonate phase from step b), and

[0024] d) The mixture obtained in step c) is spray-dried at an inlet temperature of 130 to 210°C and an outlet temperature of 50 to 130°C.

[0025] According to a further aspect of the invention, a dry, stabilized probiotic composition is provided that is obtainable by the method according to the invention.

[0026] According to a further aspect of the present application, there is provided a product comprising the dry stabilized probiotic composition according to the present application, wherein said product is a tablet, a capsule, a chewable tablet, a chewable chewing gum, a chewable pastille, a lozenge, a powder, a granule, a pellet, a paste, a cream, a food, a feed or a beverage.

[0027] According to a further aspect of the present application, there is provided the use of the dry stabilized probiotic composition according to the present application in a pharmaceutical, nutritional or cosmetic application.

[0028] Advantageous embodiments of the present application are defined in the respective dependent claims.

[0029] According to one embodiment of the present application, the probiotic composition comprises a probiotic microorganism culture selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactococcus lactis, Enterococcus faecium, Escherichia coli Nissle 1917, Escherichia coli criodesiccata (O83:K24:H31), Saccharomyces boulardii, Saccharomyces cerevisiae and mixtures thereof.

[0030] According to another embodiment of the present application, the probiotic composition comprises the probiotic microorganism culture in an amount of at least 50 wt.-%, more preferably at least 75 wt.-%, even more preferably at least 90 wt.-%, even more preferably at least 95 wt.-%, and most preferably the probiotic composition consists of the probiotic microorganism culture, based on the total weight of the probiotic composition.

[0031] According to another embodiment of the present application, the probiotic composition is a dry composition or an aqueous suspension, and preferably the probiotic composition is a dry composition.

[0032] According to another embodiment of the present application, the surface-reacted calcium carbonate has:

[0033] a volume median particle size d of 0.1 to 75 pm, preferably 0.5 to 50 pm, more preferably 1 to 40 pm, even more preferably 1.2 to 30 pm, and most preferably 1.5 to 15 pm 50 , and / or

[0034] a volume top cut particle size d of 0.2 to 150 pm, preferably 1 to 100 pm, more preferably 2 to 80 pm, even more preferably 2.4 to 60 pm, and most preferably 3 to 30 pm 98 , and / or

[0035] 15 m 2 / g to 200 m 2 / g, preferably 20 m 2 / g to 180 m 2 / g, more preferably 25 m 2 / g to 140 m 2 / g, even more preferably 27 m 2 / g to 120 m 2 / g, and most preferably 30 m 2 / g to 100 m 2 / g, and / or

[0036] a pore volume of 0.1 to 2.3 cm 3 / g, preferably 0.2 to 2.0 cm 3 / g, more preferably 0.3 to 1.8 cm 3 / g, and most preferably 0.35 to 1.6 cm 3intra-particle intruded specific pore volume in the range of 0.1 to 1 cm3 / g, which is calculated from mercury porosimetry measurements.

[0037] According to another embodiment of the present application, the natural ground calcium carbonate is selected from the group consisting of marble, chalk, limestone and mixtures thereof; or

[0038] The precipitated calcium carbonate is selected from the group consisting of precipitated calcium carbonate having an aragonite, vaterite or calcite crystal form and mixtures thereof; and / or

[0039] The at least one H3O + The counterion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, citric acid, oxalic acid, acidic salts, acetic acid, formic acid and mixtures thereof; preferably, the at least one H3O + The counterion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid, H2PO4 - which is at least partially neutralized by cations selected from Li + , Na + and / or K + , HPO4 2- which is at least partially neutralized by cations selected from Li + , Na + , K + , Mg 2+ and / or Ca 2+ , and mixtures thereof; more preferably, the at least one H3O + The counterion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid or mixtures thereof; and most preferably, the at least one H3O + The counterion donor is phosphoric acid.

[0040] According to another embodiment of the present application, the weight ratio of probiotic microorganism culture: surface-reacted calcium carbonate is in the range of 5:95 to 40:60, preferably 10:90 to 35:65, more preferably 15:85 to 30:70, and most preferably 20:80 to 25:75.

[0041] According to another embodiment of the present application, the stabilizing agent is a dry-stable agent and / or a shelf-life preservative.

[0042] According to another embodiment of the present application, the probiotic composition is a pharmaceutical probiotic composition, a nutritional probiotic composition or a cosmetic probiotic composition, and / or the probiotic composition is comprised by a tablet, a capsule, a chewable tablet, a chewable chewing gum, a chewable pastille, a lozenge, a powder, a granulate, a pellet, a paste, a cream, a food, a feed or a beverage.

[0043] According to another embodiment of the present application, the concentration of viable probiotic microorganism culture is increased by at least 5%, preferably by at least 10%, more preferably by at least 15%, and most preferably by at least 20% after drying the probiotic composition compared to a probiotic composition comprising maltodextrin as stabilizing agent.

[0044] It is to be understood that for the purposes of the present application, the following terms have the following meanings:

[0045] As used herein, the term "probiotic" refers to one or more microorganisms that confer a health benefit on the host organism (e.g. a human). Examples of health benefits derived from probiotic microorganisms are a reduction in pathogen load in the digestive tract, an improved microbial fermentation pattern in the digestive tract, an improved nutrient absorption, an improved immune function, an assisted digestion or a relief of symptoms of irritable bowel disease and colitis.

[0046] The term "microorganism" in the meaning of the present application refers to a single-celled organism, such as a bacterium or a yeast.

[0047] As used herein, the term "microorganism culture" refers to a preparation of microorganisms, which optionally comprises nutrients, microbial secretions and other soluble materials present in a microbial fermentation culture.

[0048] A "probiotic composition" in the meaning of the present application is a composition comprising a probiotic microorganism culture. For example, the probiotic microorganism culture can be present in the probiotic composition in an amount of at least 10 wt.-%, preferably in an amount of at least 20 wt.-%, more preferably in an amount of at least 30 wt.-%, and most preferably in an amount of at least 50 wt.-%, based on the total weight of the probiotic composition.

[0049] A "natural ground calcium carbonate" (GCC) in the meaning of the present application is a calcium carbonate which is obtained from natural sources (e.g. limestone, marble or chalk) and which is processed by wet and / or dry processing (e.g. grinding, sieving and / or fractionation, e.g. by a cyclone or classifier).

[0050] "Precipitated calcium carbonate" (PCC) in the sense of the present invention is a synthetic material, which is obtained by precipitation after reaction of carbon dioxide and lime in an aqueous, semi-dry or humid environment or by precipitation of calcium and carbonate ion sources in water. PCC can be in the form of vaterite, calcite or aragonite crystals. PCC is described, for example, in EP2447213 A1, EP2524898 A1, EP2371766 A1, EP1712597 A1, EP1712523 A1 or WO2013142473 A1.

[0051] The term "surface-reacted" in the sense of the present invention shall be used to indicate that the material has undergone a process which comprises treating the material in an aqueous environment with H3O + ion sources, followed by a crystallization process, which can occur in the absence or presence of further crystallization additives.

[0052] "H3O + ion sources" in the context of the present invention are Bronsted acids and / or acidic salts, i.e. salts containing acidic hydrogen. As used herein, the term "acid" refers to acids in the meaning of the Bronsted and Lowry definition (e.g. H2SO4, HSO4 – ). The term "free acid" refers only to those acids in their fully protonated form (e.g. H2SO4).

[0053] The "particle size" of a particulate material is described by its particle size distribution d x . Unless otherwise stated, the value d x represents the diameter relative to which x% by weight of the particles have a diameter less than d x . This means, for example, that a d 20 value is the particle size at which 20% by weight of all particles are smaller than this particle size. Thus, a d 50 value is the weight median particle size, i.e. 50% by weight of all particles are smaller than this particle size. For the purposes of the present invention, the particle size is designated as the weight median particle size d 50 (weight), unless otherwise stated. The particle size is determined by using a Sedigraph TM 5100 instrument of the Micromeritics Instrument Corporation or a Sedigraph TM5120 instrument. The method and instrument are known to the skilled person and are commonly used to determine the particle size of fillers and pigments. The measurement is performed in an aqueous solution of 0.1 wt% Na4P207.

[0054] For certain materials explicitly specified herein, the "particle size" is described as a volume-based particle size distribution. This is for example indicated by "volume-based median particle size", "volume median particle size" or "volume top cut particle size". The volume median particle size d 50 The evaluation is performed by using a Malvern Mastersizer 2000 or 3000 laser diffraction system. The d 50 or d 98 value indicates the diameter value below which 50% or 98% by volume, respectively, of the particles have. The raw data obtained by the measurement are analyzed by using the Mie theory, where a refractive index of the particles of 1.57 and an absorption index of 0.005 are employed. The measurement is performed in an aqueous solution of 0.1 wt% Na4P207.

[0055] The term "particle body" in the sense of the present application refers to a material composed of a plurality of particles. The plurality of particles can for example be defined by its particle size distribution. The expression "particle body material" can comprise granules, powder, fines, chips or crumbs.

[0056] The "specific surface area" (expressed in m 2 / g) used throughout this document can be determined by the Brunauer Emmett Teller (BET) method with nitrogen as adsorbing gas and by using an ASAP 2460 instrument from Micromeritics. This method is well known to the skilled person and is defined in ISO 9277:2010. Prior to such a measurement, the sample is filtered in a Büchner funnel, rinsed with deionized water and dried in an oven at 110°C for at least 12 hours. The total surface area (expressed in m 2 ) of the material can be obtained by multiplying the specific surface area (expressed in m 2 / g) of the material with the mass (expressed in g).

[0057] In the context of the present application, the term "pore" is to be understood to describe the space present between and / or within particles, i.e. it is formed by the particles when they are packed together in closest adjacent contact, e.g. in a powder or compact, and / or is the void space within a porous particle, and it allows the passage of a liquid under pressure and / or supports the absorption of a surface wetting liquid when saturated with the liquid.

[0058] The term "drying" means, unless otherwise specified, a process according to which at least a portion of the water is removed from a material to be dried, so as to reach a constant weight of the "dried" material obtained at 200°C. Furthermore, a "dried" or "dry" material can be defined by its total water content, which is, unless otherwise specified, less than or equal to 1.0 wt.%, preferably less than or equal to 0.5 wt.%, more preferably less than or equal to 0.2 wt.% and most preferably 0.03 to 0.07 wt.%, based on the total weight of the dried material.

[0059] For the purposes of the present application, "water-insoluble" materials are defined as those which, when mixed with 100 ml of deionized water and filtered at 20°C to recover a liquid filtrate, provide less than or equal to 0.1 g of recovered solid material after evaporation of 100 g of the liquid filtrate at 95 to 100°C. "Water-soluble" materials are defined as those which result in more than 0.1 g of solid material being recovered after evaporation of 100 g of the liquid filtrate at 95 to 100°C. To assess whether a material is insoluble or soluble material in the sense of the present application, the sample amount should be greater than 0.1 g, preferably 0.5 g or more.

[0060] A "suspension" or "slurry" comprises, in the sense of the present application, undissolved solids and water, and optionally further additives, and usually comprises a high amount of solids, and is thus more viscous and can have a higher density than the liquid from which it is formed.

[0061] Where the indefinite article "a" or "an" or "the" is used, including in reference to a singular noun form, this is to be understood as referring also to a plurality of said noun forms, unless otherwise indicated.

[0062] The use of the term "comprising" in the present description and claims means that other elements can also be present. For the purposes of the present application, the term "consisting of is considered to be a preferred embodiment of the term "comprising". If a group is defined to comprise at least one of a plurality of elements, and each individual element is defined specifically, then "comprising at least one of the group" refers to each individual element and also to the group as a whole.

[0063] The terms such as "obtainable" or "definable" and "obtained" or "defined" are used interchangeably. This means, for example, that unless the context clearly dictates otherwise, the term "obtained" does not imply that an embodiment must be obtained by a series of steps following the term "obtained", although such a limited interpretation is always encompassed as a preferred embodiment by the terms "obtained" or "defined".

[0064] Whenever the terms "comprising" or "having" are used, these terms are meant to be equivalent to "including" as defined herein above.

[0065] According to the present application, the use of a surface-reacted calcium carbonate as stabilizing agent for probiotic compositions is provided. The surface-reacted calcium carbonate is a reaction product of natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O + ion donors, wherein the carbon dioxide is formed in situ and / or is supplied from an external source. + The ion donor treatment is formed in situ and / or is supplied from an external source.

[0066] The details and preferred embodiments of the use according to the present application will be set out in more detail hereinafter. It is to be understood that these technical details and embodiments also apply to the method, process, composition and article according to the present application.

[0067] surface-reacted calcium carbonate

[0068] The surface-reacted calcium carbonate is a reaction product of natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O + ion donors, wherein the carbon dioxide is formed in situ and / or is supplied from an external source. + The ion donor treatment is formed in situ and / or is supplied from an external source.

[0069] H3O + The ion donors are Bronsted acids and / or acidic salts in the context of the present application.

[0070] In a preferred embodiment of the present application, the surface-reacted calcium carbonate is obtained by a method comprising the following steps: (a) providing a suspension of natural or precipitated calcium carbonate, (b) adding at least one acid having a pK a value of 0 or less at 20°C or a pK avalues, and (c) treating the suspension of step (a) with carbon dioxide before, during or after step (b). According to another embodiment, the surface-reacted calcium carbonate is obtained by a process comprising the steps of: (A) providing natural or precipitated calcium carbonate, (B) providing at least one water-soluble acid, (C) providing gaseous CO2, (D) contacting the natural or precipitated calcium carbonate of step (A) with the at least one acid of step (B) and with the CO2of step (C), characterized in that (i) the at least one acid of step B) has a pK a associated with the ionization of its first available hydrogen and forms the corresponding anion upon loss of the first available hydrogen, which is capable of forming a water-soluble calcium salt, and (ii) after contacting the at least one acid with the natural or precipitated calcium carbonate, at least one water-soluble salt is additionally provided, which in case of a hydrogen-containing salt has a pK a associated with the ionization of its first available hydrogen and the salt anion thereof is capable of forming a water-insoluble calcium salt.

[0071] The "natural ground calcium carbonate" (GCC) is preferably selected from the group of minerals comprising calcium carbonate selected from the group comprising marble, chalk, limestone and mixtures thereof. The natural calcium carbonate can comprise further naturally occurring components, such as silico-aluminates and the like.

[0072] Generally, the grinding of the natural ground calcium carbonate can be a dry or wet grinding step and can be carried out with any conventional grinding device, for example under such conditions that the comminution is mainly due to impact with a second body, i.e. in one or more of the following: ball mill, rod mill, vibration mill, roll crusher, centrifugal impact mill, vertical bead mill, attrition mill, pin mill, hammer mill, powder mill, shredder, de-lumper, knife cutter or other such devices known to the skilled person. In case the mineral material comprising calcium carbonate comprises a wet ground mineral material comprising calcium carbonate, the grinding step can be carried out under such conditions that autogenous grinding occurs and / or by horizontal ball milling and / or other such processes known to the skilled person. The thus obtained wet processed ground mineral material comprising calcium carbonate can be washed and dewatered prior to drying by well-known processes, for example by flocculation, filtration or forced evaporation. The subsequent drying step, if needed, can be carried out in a single step, for example spray drying, or in at least two steps. It is also common that such mineral materials undergo beneficiation steps (for example flotation, bleaching or magnetic separation steps) to remove impurities.

[0073] "Precipitated calcium carbonate" (PCC) in the sense of the present application is a synthetic material, which is usually obtained by precipitation after reaction of carbon dioxide and calcium hydroxide in an aqueous environment or by precipitation of calcium and carbonate ions (e.g. CaCI2and Na2CO3) from solution. A further possible way of producing PCC is the lime soda process or the Solvay process, wherein PCC is a by-product of ammonia production. Precipitated calcium carbonate exists in three main crystal forms: calcite, aragonite and vaterite, and for each of these crystal forms there are many different polymorphs (crystal habits). Calcite has a trigonal structure with typical crystal habits such as scalenohedral (S-PCC), rhombohedral (R-PCC), hexagonal prismatic, pinacoidal, colloidal (C-PCC), cubic and prismatic (P-PCC). Aragonite is an orthorhombic structure with typical crystal habits of bipyramidal crystals, various fancy shapes of thin prismatic, sickle-shaped, steep pyramidal, chisel-shaped crystals, branching trees and coral or worm-like forms. Vaterite belongs to the hexagonal crystal system. The obtained PCC slurry can be dewatered and dried mechanically.

[0074] According to one embodiment of the present application, the precipitated calcium carbonate is a precipitated calcium carbonate, which preferably comprises aragonite, vaterite or calcite mineralogical crystal form or mixtures thereof.

[0075] The precipitated calcium carbonate can be milled before treatment with carbon dioxide and at least one H3O + ion donor with the same means as described above for milling natural calcium carbonate.

[0076] According to one embodiment of the present application, the natural or precipitated calcium carbonate is in the form of particles having a weight median particle size d 50 of 0.05 to 10.0 pm, preferably 0.2 to 5.0 pm, more preferably 0.4 to 3.0 pm, most preferably 0.6 to 1.2 pm, especially 0.7 pm. According to a further embodiment of the present application, the natural or precipitated calcium carbonate is in the form of particles having a top cut particle size d 98 of 0.15 to 55 pm, preferably 1 to 40 pm, more preferably 2 to 25 pm, most preferably 3 to 15 pm, especially 4 pm.

[0077] The natural and / or precipitated calcium carbonate can be used in dry or suspended in water. Preferably, the respective slurry has a natural or precipitated calcium carbonate content in the range of 1 to 90 wt.-%, more preferably 3 to 60 wt.-%, even more preferably 5 to 40 wt.-%, and most preferably 10 to 25 wt.-%, based on the weight of the slurry.

[0078] One or more H3O used to prepare surface-reacted calcium carbonate + The ion donor can be any strong acid, moderately strong acid, or weak acid, or a mixture thereof, which generates H3O under the preparation conditions. + Ions. According to the present invention, the at least one H3O + The ion donor can also be an acidic salt, which generates H3O under the preparation conditions. + ion.

[0079] According to one embodiment, the at least one H3O + The ion donor is one with a pK value of 0 or less at 20 °C. a Strong acid.

[0080] According to another embodiment, the at least one H3O + The ion donor has a pK value of 0 to 2.5 at 20 °C. a A moderately strong acid. If pK at 20℃... a If the value is 0 or less, then the acid is preferably selected from sulfuric acid, hydrochloric acid, or mixtures thereof. If pK at 20°C... a If the value is between 0 and 2.5, then the H3O + The ion donor is preferably selected from H₂SO₃, H₃PO₄, oxalic acid, or mixtures thereof. The at least one H₂SO₄ ion donor... + Ion donors can also be acidic salts, such as HSO4. - or H2PO4 - It is at least partially contained by the corresponding cation, such as Li + Na + or K + Neutralization, or HPO4 2- It is at least partially contained by the corresponding cation, such as Li + Na + K + Mg 2+ or Ca 2+ Neutralization. The at least one H3O + Ion donors can also be mixtures of one or more acids and one or more acidic salts.

[0081] According to another embodiment, the at least one H3O + The ion donor is a weak acid with a pK value greater than 2.5 and less than or equal to 7. a The value (when measured at 20°C) is associated with the ionization of the first available hydrogen and has a corresponding anion capable of forming a water-soluble calcium salt. Subsequently, at least one additional water-soluble salt is provided, which, in the case of a hydrogen-containing salt, has a pK value greater than 7. aassociated with the ionization of the first available hydrogen and whose salt anion is capable of forming a water insoluble calcium salt. According to this preferred embodiment, the weak acid has a pK value greater than 2.5 to 5 at 20°C a and more preferably the weak acid is selected from the group consisting of acetic acid, formic acid, propionic acid and mixtures thereof. Exemplary cations of the water soluble salt are selected from the group consisting of potassium, sodium, lithium and mixtures thereof. In a more preferred embodiment, the cation is sodium or potassium. Exemplary anions of the water soluble salt are selected from the group consisting of phosphate, dihydrogen phosphate, monohydrogen phosphate, oxalate, silicate, mixtures thereof and hydrates thereof. In a more preferred embodiment, the anion is selected from the group consisting of phosphate, dihydrogen phosphate, monohydrogen phosphate, mixtures thereof and hydrates thereof. In a most preferred embodiment, the anion is selected from the group consisting of dihydrogen phosphate, monohydrogen phosphate, mixtures thereof and hydrates thereof. The water soluble salt addition can be performed dropwise or in one step. In the case of dropwise addition, the addition preferably occurs over a period of 10 minutes. More preferably, the salt is added in one step.

[0082] According to one embodiment of the present application, the at least one H3O + ion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, citric acid, oxalic acid, acetic acid, formic acid and mixtures thereof. Preferably, the at least one H3O + ion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid, H2PO4 - , which is at least partially neutralized by the corresponding cation, e.g. Li + , Na + or K + , HPO4 2- , which is at least partially neutralized by the corresponding cation, e.g. Li + , Na + , K + , Mg 2+ or Ca 2+ , and mixtures thereof; more preferably, the at least one acid is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid or mixtures thereof; and most preferably, the at least one H3O + ion donor is phosphoric acid.

[0083] The one or more H3O + ion donors can be added to the suspension as a concentrated solution or as a more dilute solution. Preferably, the H3O +The molar ratio of the H3O+donor to the natural or precipitated calcium carbonate is in the range of 0.01 to 4, more preferably 0.02 to 2, even more preferably 0.05 to 1, and most preferably 0.1 to 0.58.

[0084] As an alternative, it is also possible to add the H3O + donor to the water prior to suspending the natural or precipitated calcium carbonate.

[0085] In a next step, the natural or precipitated calcium carbonate is treated with carbon dioxide. If the H3O + donor treatment of the natural or precipitated calcium carbonate is performed using a strong acid, such as sulfuric acid or hydrochloric acid, then the carbon dioxide is formed automatically. Alternatively or in addition, the carbon dioxide can be supplied from an external source.

[0086] H3O + donor treatment and the treatment with carbon dioxide can be performed simultaneously, which is the case when a strong acid or a medium-strong acid is used. It is also possible to first perform the H3O + donor treatment, for example with a medium-strong acid having a pK a in the range of 0 to 2.5 at 20°C, wherein the carbon dioxide is formed in situ and thus the carbon dioxide treatment will automatically be performed simultaneously with the H3O + donor treatment, followed by a further treatment with carbon dioxide supplied from an external source.

[0087] In a preferred embodiment, the H3O + donor treatment step and / or the carbon dioxide treatment step is repeated at least once, more preferably several times. According to one embodiment, the at least one H3O + donor is added over a period of at least about 5 minutes, preferably at least about 10 minutes, typically about 10 to about 20 minutes, more preferably about 30 minutes, even more preferably about 45 minutes, and sometimes about 1 hour or more.

[0088] After the H3O + donor treatment and the carbon dioxide treatment, the pH of the aqueous suspension (measured at 20°C) naturally reaches a value of greater than 6.0, preferably greater than 6.5, more preferably greater than 7.0, even more preferably greater than 7.5, thereby producing the surface-reacted natural or precipitated calcium carbonate as an aqueous suspension having a pH of greater than 6.0, preferably greater than 6.5, more preferably greater than 7.0, even more preferably greater than 7.5.

[0089] In a particularly preferred embodiment, the surface-reacted calcium carbonate is a reaction product of a natural ground calcium carbonate (GNCC) with carbon dioxide and phosphoric acid, wherein the carbon dioxide is formed in situ by phosphoric acid treatment.

[0090] Further details on the preparation of surface-reacted natural calcium carbonate are disclosed in WO0039222 A1, WO2004083316 A1, WO2005121257 A2, WO2009074492 A1, EP2264108 A1, EP2264109 A1 and US20040020410 A1, the contents of which are hereby included in the present application.

[0091] Similarly, surface-reacted precipitated calcium carbonate is obtained. As can be taken from WO2009074492 A1 in detail, surface-reacted precipitated calcium carbonate is obtained by contacting precipitated calcium carbonate with H3O + ions and anions which are dissolved in an aqueous medium and which are capable of forming water-insoluble calcium salts, to form a slurry of surface-reacted precipitated calcium carbonate, wherein the surface-reacted precipitated calcium carbonate comprises insoluble, at least partially crystalline calcium salts of the anions formed on the surface of at least a portion of the precipitated calcium carbonate.

[0092] The dissolved calcium ions correspond to an excess of dissolved calcium ions over the dissolved calcium ions naturally generated upon dissolving precipitated calcium carbonate by H3O + ions, wherein the H3O + ions are provided only in the form of counterions of the anions, i.e. via addition of the anions in the form of acids or non-calcium acidic salts, and in the absence of any further calcium ions or calcium ion generating sources.

[0093] The excess of dissolved calcium ions is preferably provided by addition of soluble neutral or acidic calcium salts or by addition of acids or neutral or acidic non-calcium salts which generate soluble neutral or acidic calcium salts in situ.

[0094] The H3O + ions can be provided by addition of acids or acidic salts of the anions or by addition of acids or acidic salts which are used at the same time to provide all or part of the excess of dissolved calcium ions.

[0095] In a further preferred embodiment of the preparation of the surface-reacted natural or precipitated calcium carbonate, the natural or precipitated calcium carbonate is contacted with the one or more H3O +The ion donor and / or the carbon dioxide are reacted in the presence of at least one compound selected from the group consisting of silicates, silica, aluminium hydroxide, alkaline earth metal aluminate such as sodium or potassium aluminate, magnesium oxide, or mixtures thereof. Preferably, the at least one silicate is selected from aluminium silicate, calcium silicate or an alkaline earth metal silicate. The one or more H3O + The ion donor and / or the carbon dioxide are reacted in the presence of at least one compound selected from the group consisting of silicates, silica, aluminium hydroxide, alkaline earth metal aluminate such as sodium or potassium aluminate, magnesium oxide, or mixtures thereof. Preferably, the at least one silicate is selected from aluminium silicate, calcium silicate or an alkaline earth metal silicate. The one or more H3O

[0096] Alternatively, the silicate and / or silica and / or aluminium hydroxide and / or alkaline earth metal aluminate and / or magnesium oxide component can be added to an aqueous suspension of natural or precipitated calcium carbonate while the natural or precipitated calcium carbonate is being reacted with the one or more H3O + The reaction of the ion donor and the carbon dioxide has already started. Further details regarding the preparation of the surface-reacted natural or precipitated calcium carbonate in the presence of at least one silicate and / or silica and / or aluminium hydroxide and / or alkaline earth metal aluminate component are disclosed in WO2004083316 Al.

[0097] The surface-reacted calcium carbonate can be kept in suspension, optionally further stabilised by a dispersant. Conventional dispersants known to the skilled person can be used. Preferred dispersants comprise polyacrylic acid and / or carboxymethylcellulose.

[0098] Alternatively, the aqueous suspension described above can be dried, thereby obtaining the solid (i.e. dry, or containing so little water that it is not in liquid form) surface-reacted natural or precipitated calcium carbonate in the form of small particles or a powder.

[0099] In a preferred embodiment, the surface-reacted calcium carbonate has a specific surface area of 15 m 2 / g to 200 m 2 / g, preferably 20 m 2 / g to 180 m 2 / g, more preferably 25 m 2 / g to 140 m 2 / g, even more preferably 27 m 2 / g to 120 m 2 / g, and most preferably 30 m 2 / g to 100 m 2 / g. For example, the surface-reacted calcium carbonate has a specific surface area of 40 m 2 / g to 100 m 2BET surface area of 50 to 500 m2 / g, preferably 100 to 400 m2 / g, more preferably 150 to 350 m2 / g, and most preferably 200 to 300 m2 / g, which is measured by using nitrogen and the BET method. The BET surface area is defined in the sense of the present application as the surface area of the particles divided by the mass of the particles. As used therein, the specific surface area is measured by adsorption using the BET isotherm (ISO 9277:2010) and is expressed in m 2 / g.

[0100] It is further preferred that the surface-reacted calcium carbonate particles have a volume median particle size d 50 (volume).

[0101] It can be further preferred that the surface-reacted calcium carbonate particles have a volume top cut particle size d 98 (volume).

[0102] The value d x represents the diameter relative to which x% of the particles have a diameter smaller than d x This means that d 98 The value d 98 The value d x The value d 50 (weight) is the weight median particle size, i.e. 50% by weight of all fines are smaller than this particle size, and d 50 (volume) is the volume median particle size, i.e. 50% by volume of all fines are smaller than this particle size.

[0103] The volume median particle size d 50 is evaluated by using a Malvern Mastersizer 2000 or 3000 laser diffraction system. The d 50 or d 98 value indicates the diameter value relative to which 50% by volume or 98% by volume of the particles, respectively, have a diameter smaller than this value. The raw data obtained by the measurement are analyzed by using the Mie theory, wherein a refractive index of the particles of 1.57 and an absorption index of 0.005 are employed.

[0104] The weight median particle size was determined by sedimentation, which is the analysis of the sedimentation behaviour in a gravitational field. The measurement was performed with a Sedigraph 5100 or 5120, Micromeritics Instrument Corporation. The method and instrument are known to the skilled person and are commonly used to determine the fine particle size of fillers and pigments. The measurement was performed in an aqueous solution of 0.1 wt% Na4P207. The sample was dispersed by using a high speed stirrer and sonicated. TM 5100 or 5120, Micromeritics Instrument Corporation. The method and instrument are known to the skilled person and are commonly used to determine the fine particle size of fillers and pigments. The measurement was performed in an aqueous solution of 0.1 wt% Na4P207. The sample was dispersed by using a high speed stirrer and sonicated.

[0105] The method and instrument are known to the skilled person and are commonly used to determine the fine particle size of fillers and pigments.

[0106] The specific pore volume was measured by mercury intrusion porosimetry using a Micromeritics Autopore V 9620 mercury porosimeter with a maximum applied mercury pressure of 414 MPa (60 000 psi), equivalent to a Laplace throat diameter of 0.004 μm (~ nm). The equilibration time used at each pressure step was 20 seconds. The sample material was sealed in a 5 cm 3 The data were corrected for mercury compression, penetrometer dilation and sample material compression using Pore-Comp software (Gane, P. A. C, Kettle, J. P., Matthews, G. P. and Ridgway, C. J., "Void Space Structure of Compressible Polymer Spheres and Consolidated Calcium Carbonate Paper-Coating Formulations", Industrial and Engineering Chemistry Research, 35(5), 1996, p 1753-1764).

[0107] The total pore volume seen in the cumulative intrusion data can be separated into two regions, where the intrusion data from 214 μm down to about 1-4 μm shows the coarse packing of the sample between any agglomerate structure that is strongly contributing. Below these diameters is the fine inter-particle packing of the particles themselves. If they also have intra-particle pores, then this region appears to be bi-modal and the intra-particle pore volume is defined by taking the specific pore volume in the mercury intrusion pores that is finer than the modal turning point (i.e. finer than the bi-modal inflection point). The sum of these three regions gives the total pore volume of the powder, but is strongly dependent on the original sample compaction / deposition of the powder at the coarse pore end of the distribution.

[0108] By taking the first derivative of the cumulative intrusion curve, a pore size distribution based on the equivalent Laplace diameter is revealed, which inevitably includes pore-shielding. The differential curve clearly shows the regions of coarse agglomerate pore structure, interparticle pore region, and intra-particle pore region, if present. Knowing the range of intra-particle pore diameters, it is possible to subtract the remaining inter-particle and inter-agglomerate pore volume from the total pore volume to give the desired pore volume of only the internal pores, in terms of pore volume per unit mass (specific pore volume). Of course, the same subtraction principle applies to isolate any of the other pore size regions of interest.

[0109] Preferably, the surface-reacted calcium carbonate has an intra-particle intrusion specific pore volume in the range of 0.1 to 2.3 cm 3 / g, more preferably 0.2 to 2.0 cm 3 / g, even more preferably 0.3 to 1.8 cm 3 / g, and most preferably 0.35 to 1.6 cm 3 / g, calculated from mercury porosimetry measurements.

[0110] The intra-particle pore size of the surface-reacted calcium carbonate is preferably in the range of 0.004 to 1.6 pm, more preferably in the range of 0.005 to 1.3 pm, even more preferably 0.006 to 1.15 pm, and most preferably in the range of 0.007 to 1.0 pm, for example 0.45 to 0.60 pm, as determined by mercury porosimetry measurements.

[0111] According to one embodiment, the surface-reacted calcium carbonate has:

[0112] 0.1 to 75 pm, preferably 0.5 to 50 pm, more preferably 1 to 40 pm, even more preferably 1.2 to 30 pm, and most preferably 1.5 to 15 pm, and 50

[0113] 0.2 to 150 pm, preferably 1 to 100 pm, more preferably 2 to 80 pm, even more preferably 2.4 to 60 pm, and most preferably 3 to 30 pm, and 98

[0114] 15 m 2 / g to 200 m 2 / g, preferably 20 m 2 / g to 180 m 2 / g, more preferably 25 m 2 / g to 140 m 2 / g, even more preferably 27 m 2 ​​ / g to 120 m 2 / g, and most preferably 30 m 2 / g to 100 m 2 / g, and / or

[0115] in the range of 0.1 to 2.3 cm 3 / g, preferably 0.2 to 2.0 cm 3 / g, more preferably 0.3 to 1.8 cm 3 / g, and most preferably 0.35 to 1.6 cm 3 / g, calculated from mercury porosimetry measurements.

[0116] Additionally or alternatively, the natural ground calcium carbonate can be selected from the group consisting of marble, chalk, limestone and mixtures thereof; or

[0117] The precipitated calcium carbonate is selected from the group consisting of precipitated calcium carbonate having an aragonite, vaterite or calcite crystal form and mixtures thereof; and / or

[0118] The at least one H3O + ion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, citric acid, oxalic acid, an acidic salt, acetic acid, formic acid and mixtures thereof; preferably, the at least one H3O + ion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid, H2PO4 - which is at least partially neutralized by cations selected from Li + , Na + and / or K + , HPO4 2- which is at least partially neutralized by cations selected from Li + , Na + , K + , Mg 2+ and / or Ca 2+ , and mixtures thereof; more preferably, the at least one H3O + ion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid or mixtures thereof; and most preferably, the at least one H3O + ion donor is phosphoric acid.

[0119] According to one embodiment of the present application, the surface-reacted calcium carbonate comprises a water-insoluble, at least partially crystalline salt of the anion of the at least one acid, which is formed on the surface of the natural ground calcium carbonate or precipitated calcium carbonate. According to one embodiment, the water-insoluble, at least partially crystalline salt of the anion of the at least one acid at least partially, preferably completely, covers the surface of the natural ground calcium carbonate or precipitated calcium carbonate. Depending on the at least one acid employed, the anion can be sulfate, sulfite, phosphate, citrate, oxalate, acetate, formate and / or negative chloride.

[0120] The surface-reacted calcium carbonate can further be treated with a surface treatment agent or can remain untreated. Suitable surface treatment agents are, for example, fatty acids, aliphatic carboxylic acids, aliphatic carboxylic acid esters, mono-substituted succinic anhydrides, mono-substituted succinic acids or phosphoric acid esters. Suitable surface treatment agents and methods for preparing surface-treated filler products thereof are described, for example, in EP 2 159 258 A1, EP 2 390 285 A1, EP 2 390 280 A1, WO 2014 / 060286 A1 and WO 2014 / 128087 A1.

[0121] According to one embodiment, the surface-reacted calcium carbonate does not comprise a surface treatment layer, i.e. untreated surface-reacted calcium carbonate is used as stabilizing agent.

[0122] Probiotic composition

[0123] According to the present application, surface-reacted calcium carbonate is used as stabilizing agent for a probiotic composition.

[0124] The probiotic composition can be a dry composition or an aqueous suspension. According to one preferred embodiment, the probiotic composition is a dry composition.

[0125] The probiotic composition can comprise any probiotic microbial culture known in the art. According to one embodiment, the probiotic composition comprises a probiotic microbial culture selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactococcus lactis, Enterococcus faecium, Escherichia coli Nissle 1917, freeze-dried Escherichia coli (O83:K24:H31), Saccharomyces boulardii, Saccharomyces cerevisiae and mixtures thereof. According to one embodiment, the probiotic composition comprises one probiotic microbial culture. According to another embodiment, the probiotic composition comprises at least two probiotic microbial cultures, for example two, three, four, five, six, seven, eight, nine or ten probiotic microbial cultures.

[0126] The probiotic microbial culture can comprise additional components prior to drying, such as nutrients, bacterial secretions and other soluble materials present in microbial fermentation cultures. According to one embodiment, the probiotic microbial culture comprises the additional components in an amount of less than 20 wt.%, more preferably less than 10 wt.% of the probiotic composition.

[0127] Methods for growing microbial cultures are known to the skilled person. For example, the culture can be grown as a pure (single strain) or mixed (multiple strains) culture of the desired microorganism in a liquid medium, which can consist of proteins or protein fractions, various fermentable carbohydrates, growth stimulators, inorganic salts, buffers, etc. in sterile whole milk, skim milk, whey or other natural substrates or combinations thereof. After inoculation, the culture is allowed to develop under incubation conditions of time and temperature, which are typically optimized. Depending on the microorganism being grown, incubation times can vary from a period of 4 to 48 hours, and temperatures can vary from 15°C to 50°C. It can also be desirable to control the pH and dissolved oxygen. After satisfactory growth has been achieved, the culture in its growth medium is typically cooled to 0°C to 15°C. After fermentation, the liquid medium can be removed from the bacterial culture by any suitable method known in the art, such as centrifugation, ultrafiltration or sedimentation.

[0128] According to one embodiment, the probiotic composition comprises probiotic microbial cultures in an amount of at least 50 wt.%, more preferably in an amount of at least 75 wt.%, even more preferably in an amount of at least 90 wt.% and most preferably in an amount of at least 95 wt.%, based on the total weight of the probiotic composition. According to one embodiment, the probiotic composition consists of the probiotic microbial cultures.

[0129] The probiotic composition can comprise further additives to enhance its performance, such as vitamins, enzymes, plasticizers, colorants, flavorants, sweeteners, antioxidants, buffering agents, slip aids or mixtures thereof.

[0130] Examples of suitable vitamins are vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin, folic acid, biotin, vitamin C, vitamin D, vitamin E, vitamin K or mixtures thereof. Examples of suitable enzymes are proteolytic enzymes, such as pancreatin. Examples of suitable colorants are food colorants, riboflavin, beta-carotene or natural colorants, preferably fruit, vegetable and / or plant extracts, such as grape, black currant, aronia, carrot, beetroot, red cabbage and hibiscus. The flavorant can be selected from any suitable natural or synthetic flavorant, such as passion fruit flavor, mango flavor, pineapple flavor, cupuacu flavor, guava flavor, cocoa flavor, papaya flavor, peach flavor, apricot flavor, apple flavor, lemon flavor, grape flavor, raspberry flavor, cranberry flavor, cherry flavor or grapefruit flavor.

[0131] Examples of suitable sweeteners are carbohydrate sweeteners, preferably monosaccharides and / or disaccharides, more preferably sucrose, fructose, glucose, maltose and mixtures thereof; saccharin, cyclamate, L-aspartyl-L-phenylalanine lower alkyl ester sweeteners (e.g., aspartame); thaumatin; dihydrochalcones; cyclamates; stevioside; glycyrrhizin, synthetic alkoxyanisoles; sucralose; suosan; miracil d; monellin; sorbitol, xylitol; talin; cyclamate; substituted imidazolines; synthetic sulfnamates, such as acesulfame, acesulfame potassium and n-substituted sulfnamates; oximes, such as perilartine; peptides, such as aspartame and succanilic acid; dipeptides; amino acid-based sweeteners, such as gem-diaminoalkanes, m- aminobenzoic acids, L-amino-dicarboxylic acid alkyls and certain amides of alpha- aminodicarboxylic acids and gem-diamines; and 3-hydroxy-4-alkoxyphenyl aliphatic or heterocyclic aromatic carboxylates; erythritol; and mixtures thereof.

[0132] Examples of suitable antioxidants include tocopherols (e.g., vitamin E), ascorbic acid (e.g., vitamin C), vitamin A (e.g., beta-carotene), grape seed extract, selenium, and coenzyme Q10, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate and mixtures thereof.

[0133] Other non-limiting examples of optional components useful in the compositions of the present application include diclofenac, naproxen, aspirin, indomethacin, omeprazole, cardiac glycosides, electrolyte preparations with sodium, potassium or magnesium salts and calcium and iron preparations, bisacodyl preparations, valproic acid, 5-ASA, steroids such as hydrocortisone, budesonide, laxatives, octreotide, cisapride, anticholinergics, calcium channel blockers, 5HT3- antagonists such as ondansetron, and peptides such as insulin, solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil of theobroma; emulsifiers such as TWEENS; wetting agents such as sodium lauryl sulfate; tableting agents such as binders, antioxidants; or preservatives.

[0134] According to one embodiment, the probiotic composition does not comprise a sugar and / or a polysaccharide, preferably the probiotic composition does not comprise maltodextrin.

[0135] The inventors of the present application surprisingly found that the addition of a surface-reacted calcium carbonate to a probiotic composition can reduce the inactivation of the probiotic microbial culture during drying and storage of the probiotic composition. In particular, it was surprisingly found that a surface-reacted calcium carbonate as defined herein can minimize cell death during drying of the probiotic composition. Without being bound to any theory, it is believed that the pH of the surface-reacted calcium carbonate, which is about 7 in aqueous solution, and its low water activity have a beneficial effect on the stability of the probiotic microbial culture.

[0136] According to one embodiment, the surface-reacted calcium carbonate can be used as a dryness stabilizer and / or a shelf-life preservative. Thus, there is provided the use of a surface-reacted calcium carbonate as a dryness stabilizer and / or a shelf-life preservative, wherein the surface-reacted calcium carbonate is a natural ground calcium carbonate or a precipitated calcium carbonate which is reacted with carbon dioxide and one or more H3O + a reaction product of an ionic donor, wherein the carbon dioxide is formed in situ and / or is supplied from an external source. + The ionic donor treatment is formed in situ and / or is supplied from an external source.

[0137] According to one embodiment, the weight ratio of probiotic microbial culture : surface-reacted calcium carbonate is from 5:95 to 40:60, preferably from 10:90 to 35:65, more preferably from 15:85 to 30:70, and most preferably from 20:80 to 25:75.

[0138] The probiotic composition can have a moisture content of at least 10 6CFU / g of viable probiotic microorganism culture. Methods for determining the viability of a microorganism culture are known in the art. For example, the viability of a probiotic microorganism culture can be determined by plating the probiotic microorganism culture on a suitable medium, e.g. solidified agar on a standard size petri dish, and determining the number of colonies formed. Units of CFU (colony forming units) are used to quantify the amount of viable probiotic microorganisms in the probiotic composition.

[0139] According to one embodiment, the probiotic composition has 10 6 CFU / g to 10 14 CFU / g, preferably 10 7 CFU / g to 10 13 CFU / g, and most preferably 10 8 CFU / g to 10 12 CFU / g. According to a further embodiment, the dry probiotic composition has 10 6 CFU / g to 10 14 CFU / g, preferably 10 7 CFU / g to 10 13 CFU / g, and most preferably 10 8 CFU / g to 10 12 CFU / g of viable probiotic microorganism culture.

[0140] The concentration of viable probiotic microorganism culture is measured immediately after drying the probiotic composition or after a certain storage time. According to one embodiment, the probiotic composition has 10 6 CFU / g to 10 14 CFU / g, preferably 10 7 CFU / g to 10 13 CFU / g, and most preferably 10 8 CFU / g to 10 12 CFU / g of viable probiotic microorganism culture after drying the probiotic composition, preferably after spray drying the probiotic composition. Additionally or alternatively, the probiotic composition can have 10 6 CFU / g to 10 14 CFU / g, preferably 10 7 CFU / g to 10 13 CFU / g, and most preferably 10 8 CFU / g to 10 12 CFU / g of viable probiotic microorganism culture after storing the dry probiotic composition for 2 weeks at 30°C and 35% humidity.

[0141] The inventors of the present application surprisingly found that surface-reacted calcium carbonate, as defined herein, can provide better stabilization performance than conventionally used stabilizing agents, such as maltodextrin.

[0142] According to one embodiment, the concentration of viable probiotic microorganism culture is increased at least 5%, preferably at least 10%, more preferably at least 15%, and most preferably at least 20% after drying the probiotic composition compared to a probiotic composition comprising maltodextrin as stabilizing agent. According to one embodiment, the drying is spray drying.

[0143] According to a further aspect of the present application, a method for stabilizing a probiotic microorganism culture is provided, comprising the steps of mixing a probiotic microorganism culture with surface-reacted calcium carbonate in an aqueous medium, wherein the surface-reacted calcium carbonate is natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O + a reaction product of an ion donor, wherein the carbon dioxide is formed in situ and / or is supplied from an external source; and drying the obtained mixture. + the ion donor treatment is formed in situ and / or is supplied from an external source; and drying the obtained mixture.

[0144] The drying can be performed by any suitable method known to the skilled person. According to one embodiment, the drying is performed by spray drying, freeze drying, spray freeze drying, flash drying, fluidized bed drying, jet drying, vacuum drying or a combination thereof, preferably by spray drying, freeze drying, spray freeze drying or a combination thereof, and most preferably spray drying.

[0145] Spray drying is a well-known industrial and economical process in the art, wherein particles are formed at the same time they are dried. Dry particulate powder can be produced from wet products such as slurries or solutions by atomizing the wet product through a rotating wheel or nozzle at high speed and directing the spray of droplets into a stream of hot air (e.g. 130 to 210 °C). The atomized droplets have a very large surface area in the form of millions of micrometer-sized droplets (e.g. 10 to 200 pm), which results in a very short drying time when exposed to hot air in a drying chamber.

[0146] During freeze drying, the mixture is first frozen below the critical temperature of the formulation and then dried by sublimation in two stages under high vacuum: a first drying, during which non-bound water is removed; and a second drying, during which bound water is removed.

[0147] Spray freeze drying is essentially a combination of spray drying and freeze drying, wherein the wet product is sprayed through an atomizing nozzle into a cold gas phase of a low temperature liquid, e.g. liquid nitrogen, so that the droplets can start to freeze during their passage through the cold gas phase and are completely frozen after contact with the low temperature liquid phase. Then, the frozen droplets are dried by freeze drying.

[0148] According to a preferred embodiment, the probiotic composition is dried by spray drying. Suitable equipment and methods are known to the skilled person. For example, the spray drying step can be performed with a fluidized bed dryer. The skilled person will adjust the drying temperature and drying time accordingly. For example, the spray drying can be performed at an inlet temperature of 130 to 210°C and an outlet temperature of 50 to 130°C. According to one embodiment, the spray drying is performed at an inlet temperature of 140 to 200°C, preferably 150 to 190°C, more preferably 160 to 180°C, and most preferably about 170°C, and / or at an outlet temperature of 60 to 120°C, preferably 65 to 110°C, more preferably 70 to 100°C, even more preferably 75 to 90°C, and most preferably about 80°C.

[0149] According to a further aspect of the present application, a process for the preparation of a dry, stabilized probiotic composition is provided, wherein the process comprises the following steps:

[0150] a) providing an aqueous probiotic composition comprising at least 75 wt.-% of a probiotic microorganism culture based on the total weight of the probiotic composition,

[0151] b) providing an aqueous suspension comprising 10 to 30 wt.-% of a surface- reacted calcium carbonate based on the total weight of the aqueous suspension, wherein the surface-reacted calcium carbonate is natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O + ion donors, wherein the carbon dioxide is provided by H3O + ion donors in situ and / or from an external source,

[0152] wherein the surface-reacted calcium carbonate has a volume median particle size d 50 of 0.1 to 75 pm, and a volume top cut particle size d 98 of 0.2 to 150 pm, and a specific surface area of 15 m 2 / g to 200 m 2 / g, measured by using nitrogen and the BET method, and

[0153] wherein the weight ratio of probiotic microorganism culture : surface-reacted calcium carbonate is 5:95 to 40:60,

[0154] c) mixing the probiotic composition of step a) with the surface-reacted calcium carbonate of step b), and

[0155] d) spray drying the mixture obtained in step c) at an inlet temperature of 130 to 210 °C and an outlet temperature of 50 to 130 °C.

[0156] According to a further aspect of the present application, there is provided a dry stabilized probiotic composition obtainable by the process according to the present application. Thus, there is provided a dry stabilized probiotic composition obtained by a process comprising the steps of:

[0157] a) providing an aqueous probiotic composition comprising at least 75 wt.-% of probiotic microorganism culture based on the total weight of the probiotic composition,

[0158] b) providing an aqueous suspension comprising 10-30 wt.-% of surface-reacted calcium carbonate based on the total weight of the aqueous suspension, wherein the surface-reacted calcium carbonate is natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O + ion donors selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid or mixtures thereof, wherein the carbon dioxide is formed in situ by the H3O + ion donor treatment and / or is supplied from an external source,

[0159] wherein the surface-reacted calcium carbonate has a volume median particle size d 50 of 0.1 to 75 pm, and a volume top cut particle size d 98 of 0.2 to 150 pm, and a specific surface area of 15 m 2 / g to 200 m 2 / g, measured by using nitrogen and the BET method, and

[0160] wherein the weight ratio of probiotic microorganism culture : surface-reacted calcium carbonate is 5:95 to 40:60,

[0161] c) mixing the probiotic composition of step a) with the surface-reacted calcium carbonate of step b), and

[0162] d) spray drying the mixture obtained in step c) at an inlet temperature of 130 to 210 °C and an outlet temperature of 50 to 130 °C.

[0163] The dry stabilized probiotic composition can be in the form of a granular material, e.g. in the form of small particles, powder, fine particles, small pieces or granules. Subsequently, the dry stabilized probiotic composition can be shaped into any other suitable form. For example, the composition can be subjected to a milling process or a compression process.

[0164] The probiotic composition can be employed in a wide range of applications, and the skilled person will adjust the composition and form of the probiotic composition for the desired application. According to one embodiment, the probiotic composition is a pharmaceutical probiotic composition, a nutritional probiotic composition or a cosmetic probiotic composition. The probiotic composition can be comprised in various products. According to one embodiment, the probiotic composition is comprised by a tablet, a capsule, a chewable tablet, a chewable chewing gum, a chewable pastille, a lozenge, a powder, a granulate, a pellet, a paste, a cream, a food product, a feed product or a beverage.

[0165] According to a further aspect of the present application, there is provided a product comprising a dry stabilized probiotic composition according to the present application, wherein the product is a tablet, a capsule, a chewable tablet, a chewable chewing gum, a chewable pastille, a lozenge, a powder, a granulate, a pellet, a paste, a cream, a food product, a feed product or a beverage.

[0166] A "food product" according to the present application is any product intended for human consumption, whereas a "feed product" refers to a product intended for animal consumption. Examples of food products are yoghurt, fermented vegetables, milk powder or vitamin / mineral complexes. Examples of feed products are pet milk and dry or wet pet food. Examples of beverages are milk, fermented milk, lactic acid bacterial beverage, fermented vegetable beverage, fermented fruit beverage, plant milk beverage or fermented plant milk beverage. Non-limiting examples of plant milk that can be used in a plant milk beverage or a fermented plant milk beverage are oat milk, rice milk, soy milk, almond milk, hemp milk, coconut milk, lupin milk, pea milk, barley milk or hazelnut milk. Examples of cosmetic probiotic compositions are probiotic skin creams, probiotic creams, probiotic gels, probiotic serums, probiotic shower gels, probiotic soaps, probiotic shampoos, probiotic face washes, probiotic masks or probiotic skin sprays.

[0167] According to a further aspect of the present application, there is provided the use of a dry stabilized probiotic composition according to the present application in a pharmaceutical, nutritional or cosmetic application.

[0168] According to one embodiment, there is provided the use of a surface-reacted calcium carbonate as a stabilizing agent for a probiotic composition, wherein the surface-reacted calcium carbonate is natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O + a reaction product of an acid donor, wherein the carbon dioxide is provided by H3O +Ion donor treatment is performed in situ and / or supplied from external sources.

[0169] The surface-reacted calcium carbonate described herein has a median particle size d of 1.2 to 30 μm, preferably 1.5 to 15 μm. 50 and a volumetric top-cut particle size d of 2.4 to 60 μm, preferably 3 to 30 μm. 98 , and 27m 2 / g to 120m 2 / g, preferably 30m 2 / g to 100m 2 The specific surface area per g (which was measured using nitrogen and the BET method), and in the range of 0.35 to 1.6 cm². 3 The intraparticle invasive pore volume within the range of / g (which is calculated from mercury porosimetry measurements).

[0170] The probiotic composition comprises a probiotic microbial culture selected from the group consisting of: Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus casei, Lactobacillus leucovorin, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactococcus lactis, Enterococcus faecalis, Escherichia coli Nissle 1917, freeze-dried Escherichia coli (O83:K24:H31), Saccharomyces boulardii, Saccharomyces cerevisiae, and mixtures thereof, and the weight ratio of probiotic microbial culture to surface-reacted calcium carbonate is 5:95 to 40:60, preferably 10:90 to 35:65, more preferably 15:85 to 30:70, and most preferably 20:80 to 25:75.

[0171] According to a further embodiment of the present invention, a method for preparing a dry, stabilized prebiotic composition is provided, wherein the method comprises the following steps:

[0172] a) Provides an aqueous probiotic composition comprising at least 75% by weight of a probiotic microbial culture based on the total weight of the probiotic composition.

[0173] b) Provide an aqueous suspension comprising 10-30% by weight of surface-reacted calcium carbonate based on the total weight of the aqueous suspension, wherein the surface-reacted calcium carbonate is naturally milled calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O selected from the group consisting of phosphoric acid. + The reaction products of the ion donor, wherein the carbon dioxide is passed through H3O + Ion donor treatment is performed in situ.

[0174] wherein the surface-reacted calcium carbonate has a volume median particle size d 50 of 1.2 to 30 pm, preferably 1.5 to 15 pm, a volume top cut particle size d 98 of 2.4 to 60 pm, preferably 3 to 30 pm, a specific surface area (measured by using nitrogen and the BET method) of 27 m 2 / g to 120 m 2 / g, preferably 30 m 2 / g to 100 m 2 / g, and an intruded specific pore volume (calculated from mercury porosimetry measurements) in the range of 0.35 to 1.6 cm 3 / g, and wherein the weight ratio of probiotic microorganism culture : surface-reacted calcium carbonate is 5 : 95 to 40 : 60,

[0175] c) mixing the probiotic composition of step a) with the surface-reacted calcium carbonate of step b), and

[0176] d) spray drying the mixture obtained in step c) at an inlet temperature of 160 to 180 °C and an outlet temperature of 70 to 90 °C.

[0177] The scope and benefits of the present application will be better understood based on the following figures and examples, which are intended to illustrate certain embodiments of the present application and are non-limiting. Examples

[0178] 1. Method

[0179] The measurement methods carried out in the examples are described below.

[0180] Particle size distribution

[0181] Volume determined median particle size d 50 (volume) and volume determined top cut particle size d 98 (volume) were evaluated by using a Malvern Mastersizer 3000 laser diffraction system (Malvern Instruments Plc., Great Britain). d 50 (volume) or d 98The (volume) values indicate the diameter value below which 50 or 98 volume percent, respectively, of the particles have a diameter. The raw data obtained by the measurement are analyzed by using the Mie theory with a refractive index of the particles of 1.57 and an absorption index of 0.005. The method and the instrument are known to the skilled person and are commonly used for determining the particle size distribution of fillers and pigments. The measurement is performed in an aqueous solution of 0.1 wt% Na4P207. The sample is dispersed by using a high speed stirrer and is sonicated.

[0182] Weight median particle size d 50 (weight) is determined by sedimentation, which is the analysis of the sedimentation behavior in the gravimetric field. The measurement is performed with a Sedigraph 5120, Micromeritics Instrument Corporation. The method and the instrument are known to the skilled person and are commonly used for determining the fine particle size of fillers and pigments. The measurement is performed in an aqueous solution of 0.1 wt% Na4P207. The sample is dispersed by using a high speed stirrer and is sonicated. TM 5120, Micromeritics Instrument Corporation. The method and the instrument are known to the skilled person and are commonly used for determining the fine particle size of fillers and pigments. The measurement is performed in an aqueous solution of 0.1 wt% Na4P207. The sample is dispersed by using a high speed stirrer and is sonicated.

[0183] The method and the instrument are known to the skilled person and are commonly used for determining the fine particle size of fillers and pigments.

[0184] Specific surface area (SSA)

[0185] The specific surface area is measured via the BET method according to ISO 9277:2010 using nitrogen after conditioning the sample by heating at 250 °C for a period of 30 minutes. Prior to such measurement, the sample is filtered in a Buchner funnel, rinsed with deionized water and dried in an oven at 110 °C for at least 12 hours.

[0186] Intraparticle intrusion specific pore volume (in cm3 / g) 3 / g)

[0187] The specific pore volume is measured by using mercury intrusion porosimetry measurements with a Micromeritics Autopore V 9620 mercury porosimeter with a maximum applied mercury pressure of 414 MPa (60 000 psi), equivalent to a Laplace throat diameter of 0.004 pm (~ nm). The equilibration time used at each pressure step was 20 seconds. The sample material was sealed in a 5 cm 3The mercury intrusion data was corrected for mercury compression, penetrometer dilatation and sample material compression using Pore-Comp software (Gane, P.A.C., Kettle, J.P., Matthews, G.P. and Ridgway, C.J., "Void Space Structure of Compressible Polymer Spheres and Consolidated Calcium Carbonate Paper-Coating Formulations", Industrial and Engineering Chemistry Research, 35(5), 1996, p1753-1764).

[0188] The total pore volume seen in the cumulative intrusion data can be separated into two regions, where the intrusion data falls from 214 μm down to about 1-4 μm, which shows the coarse packing of the sample between any agglomerate structures that are strongly contributing. Below these diameters is the fine inter-particle packing of the particles themselves. If they also have intra-particle pores, then this region appears to be bi-modal, and the intra-particle pore volume is defined by taking the specific pore volume from the mercury intrusion into the pores that is finer than the modal turning point (i.e. finer than the bi-modal inflection point). The sum of these three regions gives the total pore volume of the powder, but is strongly dependent on the original sample compaction / deposition of the powder at the coarse pore end of the distribution.

[0189] By taking the first derivative of the cumulative intrusion curve, the pore size distribution based on the equivalent Laplace diameter is revealed, which inevitably includes pore-shielding. The differential curve clearly shows the coarse agglomerate pore structure region, the inter-particle pore region and the intra-particle pore region, if present. Knowing the intra-particle pore diameter range, it is possible to subtract the remaining inter-particle and inter-agglomerate pore volume from the total pore volume to give the desired intra-pore pore volume, in terms of pore volume per unit mass (specific pore volume). Of course, the same subtraction principle applies to isolate any of the other pore size regions of interest.

[0190] 2. Materials

[0191] Stabilizing agent

[0192] SRCC: surface-reacted calcium carbonate (d 50 (Volume) = 6.6 μm, d 98 (Volume) = 13.7 μm, SSA = 59.9 m 2 (Volume) = 6.6 μm, d 3 / g (for a pore diameter range of 0.004 to 0.51 μm).

[0193] SRCC was obtained by preparing a 350 liter aqueous suspension of ground calcium carbonate in a mixing vessel by adjusting the solid content of ground limestone calcium carbonate from Omya SAS, Orgon, having a weight-based median particle size d 50 (weight) of 10 wt% based on the total weight of the aqueous suspension.

[0194] While mixing the slurry at a speed of 6.2 m / s, 11.2 kg of phosphoric acid was added to the suspension in the form of an aqueous solution containing 30 wt% phosphoric acid over a period of 20 minutes at a temperature of 70°C. After the addition of the acid, the slurry was stirred for another 5 minutes, after which it was removed from the vessel and dried using a jet dryer.

[0195] Maltodextrin (comparative stabilizer).

[0196] Probiotic microorganism

[0197] Lactobacillus plantarum WCFS1.

[0198] 3. Example 1

[0199] 3.1. Fermentation and harvesting of probiotic bacteria

[0200] First, a pre-inoculum was prepared from Lactobacillus plantarum strain in 5 mL of MRS-B medium. This was incubated at 37°C for about 10 hours. After that, the actual fermentation was inoculated with 1% of the pre-inoculum. The fermentation medium used was also MRS-B, which was then incubated at a temperature of 37°C overnight (~ 16-17 hours). After fermentation, samples were taken for CFU determination.

[0201] For harvesting, the completed fermentation was centrifuged at 5000 rpm for 15 minutes (4°C). The resulting granular precipitate was resuspended in 100 mL of PBS (phosphate buffered saline) buffer and divided into 2 equal sized parts. The concentration of probiotic microorganisms in the suspension was 4.2 x 10 10 CFU / g. Each of these parts was mixed with 450 g of a stabilizer solution containing 20% (w / w) stabilizer, resulting in 2 solutions of ~ 500 g. One of the stabilizer solutions contained maltodextrin, while the other contained the stabilizing agent of the present invention. Note that both stabilizer solutions were sterilized (15 minutes at 121°C) before mixing with the resuspended granular precipitate parts.

[0202] 3.2. Spray drying and shelf life test

[0203] Before spray drying, a sample of each stabilizer solution was taken for CFU determination before drying. CFU analysis was performed according to ISO 11133-1 :2009.

[0204] For the spray drying test, a Büchi benchtop spray drying system was used. An inlet temperature of 170 °C and an outlet temperature of 80 °C were used to spray dry all materials. Again, the resulting powder was sampled for CFU analysis for yield determination after drying.

[0205] The remaining powder was used for shelf life analysis. For each stabilizer powder, 0.5-1 g was dosed in a reactor tube with filter cap. Thereby, these tubes were incubated at 30 °C with a humidity level of 35% horizontally (for greater air contact area) for 2 weeks. After this incubation period, again sampling was done for CFU analysis.

[0206] 3.3. Results

[0207] The results about CFU measurements at different steps in the benchmark study are shown in Table 1. It can be seen that the concentration of viable probiotic microorganisms (CFU / g) of the spray dried powder decreases from before spray drying until the end of the shelf life study. The amount after fermentation seems slightly lower than before spray drying, but this is only due to the dry matter approximation for the back-calculation of this value. In addition, the standard deviation is relatively high for each of the measurements (as is usually the case for CFU determinations). These numbers about the standard deviation are shown in Table 2.

[0208] About the product properties, the maltodextrin solution before spray drying is clear, while the suspension of the stabilizing agent of the invention is turbid (milky). The maltodextrin powder is more difficult to resuspend than the mineral stabilizer powder, although the latter tends to sediment. The density of the maltodextrin powder is also much larger than for the mineral stabilizer powder, with a bulk density of around 470 g / L compared to about 200 g / L for the mineral stabilizer powder.

[0209] Table 1: CFU / gram powder for each step in the benchmark study. The concentration after fermentation is back-calculated based on the approximate dry matter.

[0210]

[0211] Table 2: Standard deviation for CFU determination for each step in the benchmark study

[0212]

[0213] The results compiled in Tables 1 and 2 show that the concentration of viable probiotic microorganisms is significantly higher in the samples of the present application after spray drying, and that the samples of the present application exhibit a significantly increased shelf life.

[0214] 4. Example 2

[0215] 4.1. Fermentation and harvesting of probiotic bacteria

[0216] First, a pre-inoculum was prepared from Lactobacillus plantarum strain in 5 mL of MRS-B medium. This was incubated at 37°C for approximately 10 hours. After that, the actual fermentation was inoculated using 1% of the pre-inoculum. The fermentation medium used was also MRS-B, which was then incubated at a temperature of 37°C overnight (~ 16-17 hours). After fermentation, samples were taken for CFU determination.

[0217] For harvesting, the completed fermentation was centrifuged at 5000 rpm for 15 minutes (4°C). The resulting granular precipitate was resuspended in 250 mL of PBS (Phosphate Buffered Saline) buffer and divided into five equal parts. Three of these parts were mixed with 450 g of a stabilizer solution containing the following ingredients (based on the total weight of the final mixture):

[0218] Stabilizer solution 1 : PBS only (control buffer solution);

[0219] Stabilizer solution 2: 5% by weight of maltodextrin;

[0220] Stabilizer solution 3: 5% by weight of SRCC.

[0221] This resulted in three 500 g sample solutions. The stabilizer solutions were sterilized (15 minutes at 121°C) before being mixed with the resuspended granular precipitate parts. The composition of the sample solutions is indicated in Table 3 below.

[0222] Table 3: Composition of sample solutions produced according to Example 2

[0223] Sample solution Amount of PBS [g] Stabilizing agent Biomass [g] Total solids [wt%] 1 4.95 -- ～5 1.99 2 0.494 5 g maltodextrin ～5 2.10 3 0.494 5 g SRCC ～5 2.10

[0224] 4.2. Spray drying

[0225] Before spray drying, a sample of each sample solution was taken for CFU determination as a reference. Spray drying was performed using a Büchi benchtop dryer.

[0226] For phase 1, all sample solutions were spray dried using an inlet temperature of 200°C and an outlet temperature of 100°C. Since the main aim of phase 1 was to obtain observable yield differences between the different sample solutions, the inlet and outlet temperatures were set relatively high to ensure greater inactivation and thus greater contrast between the samples.

[0227] During phase 2, the drying conditions were: 180°C and 80°C for the inlet and outlet temperatures, respectively. The main aim of phase 2 was to determine the effect of stabilisers and formulations on survival through the gut and shelf life. Therefore, relatively mild spray drying conditions were chosen to maximise the starting CFU for the tests that subsequently took place.

[0228] 4.3. Formulations (phase 2)

[0229] Three different probiotic formulations were prepared to further evaluate the possible applications of the stabilising agents of the present application, using spray-dried sample solutions 2 and 3, which contained the stabilising agents maltodextrin and SRCC, respectively, and were subjected to digestion and / or shelf life experiments. For the different formulations, the powder obtained by spray drying of the respective sample solution according to the phase 2 conditions was mixed with the specific bulking components shown below in a 1 : 1 weight ratio.

[0230] Milk powder for liquid intake

[0231] The spray-dried powder was mixed with standard skimmed milk powder in a 1 : 1 weight ratio. A portion of this dry blend was used for shelf life analysis (dry), while another portion was dissolved for digestion studies.

[0232] Tablet for intake

[0233] The spray-dried powder was mixed with lactose powder in a 1 : 1 weight ratio. The powder blend was then manually pressed into tablets of ~1 gram. These tablets were subjected to digestion studies and shelf life experiments.

[0234] Cream for skin application

[0235] A base cream was prepared by mixing oleyl alcohol with cetyl alcohol in a 1 : 1 weight ratio, resulting in a vaseline-like cream. The cream was then mixed with the spray-dried powder in a 1 : 1 weight ratio, after which it was subjected to shelf life analysis.

[0236] 4.4. Digestion studies and shelf life experiments

[0237] In vitro digestion test

[0238] An in vitro digestion model was used to evaluate the intestinal survival of Lactobacillus plantarum WCFS1, using different stabilizers and formulations. This model has been validated to show strain-specific GI persistence comparable to in vivo methods (Van Bokhorst-van de Veen et al., 2012).

[0239] Digestion studies were performed in an in vitro setup, in which a glass sample holder was used in a shaking water bath to mimic intestinal mixing. Initially, 3 grams of a formulation (dry blend of milk powder or tablets) was added to 27 g sterile water in the sample holder (resulting in a 10 wt% solution). This was done to mimic the actual intake of a typical milk powder solution and tablet, respectively, with water. Four resulting formulations were subjected to an in vitro digestion protocol as presented in Figure 1 , in which the times of addition of different digestion fluids and setting the pH are indicated in minutes. The in vitro digestion protocol comprised the following steps:

[0240] -0 min: addition of 2 mL of a saliva buffer, comprising alpha-amylase;

[0241] -5 min: pH adjustment to 2 using a 0.5 M HC1 solution in 15 min;

[0242] -20 min: addition of 8 mL of a gastric buffer, comprising lipase and pepsin;

[0243] -80 min: taking 0.3 mL sample (after gastric phase) and start of pH adjustment to 6 using 1 M NaOH in 5 min;

[0244] -85 min: addition of 10 mL of an intestinal buffer, comprising pancreatic enzyme preparation and bile;

[0245] -175 min: taking 0.3 mL sample (after intestinal phase) and end of experiment.

[0246] The taken samples were then used for CFU analysis.

[0247] Shelf life test

[0248] For shelf-life experiments, the same formulations as used for the digestion studies were evaluated, as well as a cream for skin application. For each formulation, 0.5-1 g was dosed in reactor tubes with filter caps. These tubes were incubated at 30 °C at a humidity level of 35% horizontally (for greater air contact area) for 2 weeks. After this incubation period, samples were taken for CFU analysis.

[0249] 4.5. Results

[0250] Spray drying

[0251] After spray drying, CFU analysis of the buffer solution (control sample) and the stabilized samples was performed according to ISO 1133-1 :2009. The results are compiled in Figure 2 Table 1. In literature, a comparison in terms of % recovery is commonly applied (Perdana et al., 2013). As can be seen from Figure 2 Table 1, the CFU values for the inventive samples comprising SRCC are always higher than the CFU values for the comparative samples comprising maltodextrin. The yield for the maltodextrin samples is in line with the literature values of 5-10% (Siemons et al., 2021), while the yield for the control sample with buffer is relatively high compared to maltodextrin.

[0252] Further, there is less material on the wall of the Buchi for the inventive samples comprising SRCC compared to the samples comprising maltodextrin. The spray-dried maltodextrin samples also have more powder clumps, which are less easily dispersed into loose powder.

[0253] In vitro digestion test

[0254] CFU analysis of the stabilized samples was performed according to ISO 1133-1 :2009 before and after the in-vitro digestion test. Figure 3 The results are shown in Table 2. In this case, the difference in performance for the sample comprising the inventive stabilization agent SRCC and the comparative sample comprising maltodextrin is striking. The inventive sample with SRCC retains 10% viability, i.e. a log 1 reduction after digestion. In contrast, the comparative sample with maltodextrin is more sensitive, where only 0.01% viability remains, i.e. a log 4 reduction after digestion. The inventive stabilization agent SRCC outperforms maltodextrin in both formulations, i.e. milk powder as well as lactose tablets.

[0255] In addition, it was observed that the milk powder formulation performs slightly better than the lactose tablet when comparing the final CFU values. A reason for this can be the fact that milk is a rich medium with both buffering capacity and nutritional content.

[0256] Shelf life test

[0257] CFU analysis of the stabilized samples was performed according to ISO 1133-1 :2009 before and after the shelf-life test. Figure 4The results of the accelerated shelf life experiment are shown. In line with the results of the in vitro digestion test, the samples of the application with SRCC outperformed the comparative samples with maltodextrin for both the milk powder formulation and the lactose tablet. In addition, the cream formulation comprising the stabilizing agent SRCC of the application performed slightly better than the cream formulation comprising the comparative stabilizing agent maltodextrin.

[0258] In summary, it has been shown that the stabilizing agent of the application is an effective stabilizing agent in the production (spray drying), delivery (shelf life) and application (in vitro digestion) of probiotic compositions. In addition, the stabilizing agent of the application outperforms the comparative stabilizing agent maltodextrin.

Claims

1. Use of surface-reacted calcium carbonate as stabilizing agent for a probiotic composition, wherein the stabilizing agent is a dryness stabilizer and / or a shelf-life preservative, wherein the surface-reacted calcium carbonate is natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O + reaction products of the ion donors, wherein the carbon dioxide is provided by H3O + ion donor treatment is formed in situ and / or supplied from an external source, wherein the surface-reacted calcium carbonate has a volume median particle size d 50 of 0.1 to 75 pm, a volume top cut particle size d 98 of 0.2 to 150 pm, and a specific surface area of 15 m 2 / g to 200 m 2 / g, measured by using nitrogen and the BET method, wherein the probiotic composition comprises a probiotic microorganism culture selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactococcus lactis, Enterococcus faecium, Escherichia coli Nissle 1917, Escherichia coli criodesiccata with O83:K24:H31 on its surface, Saccharomyces boulardii, Saccharomyces cerevisiae and mixtures thereof, and wherein the weight ratio of probiotic microorganism culture : surface-reacted calcium carbonate is from 5 : 95 to 40 :

60.

2. The use of claim 1, wherein the probiotic composition comprises the probiotic microorganism culture in an amount of at least 50 wt.-%, based on the total weight of the probiotic composition.

3. The use of claim 2, wherein the probiotic composition comprises the probiotic microorganism culture in an amount of at least 75 wt.-%, based on the total weight of the probiotic composition.

4. The use of claim 2, wherein the probiotic composition comprises the probiotic microorganism culture in an amount of at least 90 wt.-%, based on the total weight of the probiotic composition.

5. The use of claim 2, wherein the probiotic composition comprises the probiotic microorganism culture in an amount of at least 95 wt.-%, based on the total weight of the probiotic composition.

6. The use of claim 2, wherein the probiotic composition consists of the probiotic microorganism culture.

7. The use of claim 1, wherein the probiotic composition is a dry composition or an aqueous suspension.

8. The use of claim 7, wherein the probiotic composition is a dry composition.

9. The use of claim 1, wherein the surface-reacted calcium carbonate has an intraparticle invasive specific pore volume in the range of 0.1 to 2.3 cm3 / g, calculated from mercury porosimetry measurements. 3 / g.

10. The use of claim 9, wherein the surface-reacted calcium carbonate has a volume median particle size d 50 50 μm.

11. The use of claim 9, wherein the surface-reacted calcium carbonate has a volume median particle size d 50 .

12. The use of claim 9, wherein the surface-reacted calcium carbonate has a volume median particle size d 50 .

13. The use of claim 9, wherein the surface-reacted calcium carbonate has a volume median particle size d 50 . of 1.5 to 15 μm.

14. The use of claim 9, wherein the surface-reacted calcium carbonate has a volume top cut particle size d of 1 to 100 μm 98 .

15. The use of claim 9, wherein the surface-reacted calcium carbonate has a volume top cut particle size d of from 2 to 80 μm 98 .

16. The use of claim 9, wherein the surface-reacted calcium carbonate has a volume top cut particle size d 98 .

17. The use of claim 9, wherein the surface-reacted calcium carbonate has a volume top cut particle size d 98 .

18. The use of claim 9, wherein the surface-reacted calcium carbonate has a specific surface area of 20 m2 / g to 180 m2 / g. 2 / g to 180 m 2 / g.

19. The use of claim 9, wherein the surface-reacted calcium carbonate has a specific surface area of 25 m 2 / g to 140 m 2 / g.

20. The use of claim 9, wherein the surface-reacted calcium carbonate has a specific surface area of 27 m 2 / g to 120 m 2 / g.

21. The use of claim 9, wherein the surface-reacted calcium carbonate has a specific surface area of 30 m 2 / g to 100 m 2 / g.

22. The use of claim 9, wherein the surface-reacted calcium carbonate has an intraparticle invasive specific pore volume in the range of 0.2 to 2.0 cm3 / g. 3 / g.

23. The use of claim 9, wherein the surface-reacted calcium carbonate has an intraparticle invasive specific pore volume in the range of 0.3 to 1.8 cm3 / g. 3 / g.

24. The use of claim 9, wherein the surface-reacted calcium carbonate has an intraparticle invasive specific pore volume in the range of 0.35 to 1.6 cm3 / g. 3 / g.

25. The use of claim 1, wherein the natural ground calcium carbonate is selected from the group consisting of marble, chalk, limestone and mixtures thereof; or the precipitated calcium carbonate is selected from the group consisting of precipitated calcium carbonate having aragonite, vaterite or calcite crystal form and mixtures thereof; and / or said H3O + the counterion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, citric acid, oxalic acid, an acidic salt, acetic acid, formic acid, and mixtures thereof.

26. The use of claim 25, wherein the H30+ + The counterion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid, H2PO4 - , HPO4 2- and mixtures thereof; the H2PO4 - is at least partially neutralized by a cation selected from Li + , Na + and / or K + and the HPO4 2- is at least partially neutralized by a cation selected from Li + , Na + , K + , Mg 2+ and / or Ca 2+ .

27. The use of claim 25, wherein the H30+ + The counterion donor is selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid or mixtures thereof.

28. The use of claim 25, wherein the H30+ + The counterion is phosphoric acid.

29. The use of claim 1, wherein the weight ratio of probiotic microorganism culture : surface-reacted calcium carbonate is from 10:90 to 35:

65.

30. The use of claim 1, wherein the weight ratio of probiotic microorganism culture : surface-reacted calcium carbonate is from 15:85 to 30:

70.

31. The use of claim 1, wherein the weight ratio of probiotic microorganism culture : surface-reacted calcium carbonate is from 20:80 to 25:

75.

32. The use of claim 1, wherein the probiotic composition is a pharmaceutical probiotic composition, a nutritional probiotic composition or a cosmetic probiotic composition, and / or the probiotic composition is comprised by a tablet, a capsule, a chewable lozenge, a powder, a granule, a pellet, a paste, a cream, a food, a feed or a beverage.

33. The use of claim 1, wherein the probiotic composition is a pharmaceutical probiotic composition, a nutritional probiotic composition or a cosmetic probiotic composition, and / or the probiotic composition is comprised by a chewable tablet, a chewable chewing gum or a lozenge.

34. The use of claim 1, wherein the concentration of viable probiotic microorganism culture is increased by at least 5% after drying the probiotic composition compared to a probiotic composition comprising maltodextrin as stabilizing agent.

35. The use of claim 34, wherein the concentration of viable probiotic microorganism culture is increased by at least 10% after drying the probiotic composition compared to a probiotic composition comprising maltodextrin as stabilizing agent.

36. The use of claim 34, wherein the concentration of viable probiotic microorganism culture is increased by at least 15% after drying the probiotic composition compared to a probiotic composition comprising maltodextrin as stabilizing agent.

37. The use of claim 34, wherein the concentration of viable probiotic microorganism culture is increased by at least 20% after drying the probiotic composition compared to a probiotic composition comprising maltodextrin as stabilizing agent.

38. A method for stabilizing a probiotic microorganism culture, comprising the steps of: mixing a probiotic microorganism culture with a surface-reacted calcium carbonate in an aqueous medium, wherein the surface-reacted calcium carbonate is natural ground calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O + a reaction product of a carbon dioxide donor, wherein the carbon dioxide is provided by H3O + the carbon dioxide donor treatment is formed in situ and / or supplied from an external source, and drying the obtained mixture, wherein the surface-reacted calcium carbonate has a volume median particle size d 50 of 0.1 to 75 pm, a volume top cut particle size d 98 of 0.2 to 150 pm, and a specific surface area of 15 m 2 / g to 200 m 2 / g, measured by using nitrogen and the BET method, wherein the weight ratio of probiotic microorganism culture : surface-reacted calcium carbonate is from 5:95 to 40:60, and 39. The method of claim 38, wherein the surface-reacted calcium carbonate is a natural ground calcium carbonate or a precipitated calcium carbonate. wherein the probiotic microorganism culture is selected from the group consisting of Bifidobacterium adolescentis, Bifidobacterium lactis, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactococcus lactis, Enterococcus faecium, Escherichia coli Nissle 1917, Escherichia coli criodesiccata with O83:K24:H31 on its surface, Saccharomyces boulardii, Saccharomyces cerevisiae and mixtures thereof.

39. The method of claim 38, wherein the drying is performed by spray drying, freeze drying, spray freeze drying, flash drying, fluid bed drying, jet drying, vacuum drying or a combination thereof.

40. A method for preparing a dry, stabilized probiotic composition, comprising the steps of: a) providing an aqueous probiotic composition comprising at least 75 wt.-% of probiotic microorganism culture based on the total weight of the probiotic composition, b) Provide an aqueous suspension comprising 10-30% by weight of surface-reacted calcium carbonate based on the total weight of the aqueous suspension, wherein the surface-reacted calcium carbonate is naturally milled calcium carbonate or precipitated calcium carbonate with carbon dioxide and one or more H3O selected from the group consisting of hydrochloric acid, sulfuric acid, sulfurous acid, phosphoric acid, oxalic acid or mixtures thereof. + The reaction products of the ion donor, wherein the carbon dioxide is passed through H3O + Ion donor treatment is performed in situ and / or supplied from external sources. The surface-reacted calcium carbonate described herein has a median particle size d of 0.1 to 75 μm. 50 Volumetric top-cutting particle size d from 0.2 to 150 μm 98 and 15 m 2 / g to 200 m 2 The specific surface area per g was measured using nitrogen and the BET method, and wherein the weight ratio of probiotic microorganism culture : surface-reacted calcium carbonate is from 5 : 95 to 40 : 60, c) mixing the probiotic composition of step a) with the surface-reacted calcium carbonate of step b), and d) spray drying the mixture obtained in step c) at an inlet temperature of from 130 to 210 °C and an outlet temperature of from 50 to 130 °C.

41. A dry, stabilized probiotic composition obtainable by the method according to claim 40.

42. A product comprising the dry, stabilized probiotic composition according to claim 41, wherein the product is a tablet, a capsule, a chewable lozenge, a powder, a granulate, a pellet, a paste, a cream, a food, a feed or a beverage.

43. A product comprising the dry, stabilized probiotic composition according to claim 41, wherein the product is a chewable tablet, a chewable chewing gum or a lozenge.

44. Use of the dry, stabilized probiotic composition according to claim 41 in a pharmaceutical, nutritional or cosmetic application, wherein the use is not a method of treatment of a disease.

Images